Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro target cell infection is characterized by the expression of the latency-associated genes ORF 73 (LANA-1), ORF 72, and K13 and by the transient expression of a very limited number of lytic genes such as lytic cycle switch gene ORF 50 (RTA) and the immediate early (IE) lytic K5, K8, and v-IRF2 genes. During the early stages of infection, several overlapping multistep complex events precede the initiation of viral gene expression. KSHV envelope glycoprotein gB induces the FAK-Src-PI3K-RhoGTPase (where FAK is focal adhesion kinase) signaling pathway. As early as 5 min postinfection (p.i.), KSHV induced the extracellular signal-regulated kinase 1 and 2 (ERK1/2) via the PI3K-PKCzeta-MEK pathway. In addition, KSHV modulated the transcription of several host genes of primary human dermal microvascular endothelial cells (HMVEC-d) and fibroblast (HFF) cells by 2 h and 4 h p.i. Neutralization of virus entry and infection by PI-3K and other cellular tyrosine kinase inhibitors suggested a critical role for signaling molecules in KSHV infection of target cells. Here we investigated the induction of ERK1/2 by KSHV and KSHV envelope glycoproteins gB and gpK8.1A and the role of induced ERK in viral and host gene expression. Early during infection, significant ERK1/2 induction was observed even with low multiplicity of infection of live and UV-inactivated KSHV in serum-starved cells as well as in the presence of serum. Entry of UV-inactivated virus and the absence of viral gene expression suggested that ERK1/2 induction is mediated by the initial signal cascade induced by KSHV binding and entry. Purified soluble gpK8.1A induced the MEK1/2 dependent ERK1/2 but not ERK5 and p38 mitogen-activated protein kinase (MAPK) in HMVEC-d and HFF. Moderate ERK induction with soluble gB was seen only in HMVEC-d. Preincubation of gpK8.1A with heparin or anti-gpK8.1A antibodies inhibited the ERK induction. U0126, a selective inhibitor for MEK/ERK blocked the gpK8.1A- and KSHV-induced ERK activation. ERK1/2 inhibition did not block viral DNA internalization and had no significant effect on nuclear delivery of KSHV DNA during de novo infection. Analyses of viral gene expression by quantitative real-time reverse transcriptase PCR revealed that pretreatment of cells with U0126 for 1 h and during the 2-h infection with KSHV significantly inhibited the expression of ORF 73, ORF 50 (RTA), and the IE-K8 and v-IRF2 genes. However, the expression of lytic IE-K5 gene was not affected significantly. Expression of ORF 73 in BCBL-1 cells was also significantly inhibited by preincubation with U0126. Inhibition of ERK1/2 also inhibited the transcription of some of the vital host genes such as DUSP5 (dual specificity phosphatase 5), ICAM-1 (intercellular adhesion molecule 1), heparin binding epidermal growth factor, and vascular endothelial growth factor that were up-regulated early during KSHV infection. Several MAPK-regulated host transcription factors such as c-Jun, STAT1alpha, MEF2, c-Myc, ATF-2 and c-Fos were induced early during infection, and ERK inhibition significantly blocked the c-Fos, c-Jun, c-Myc, and STAT1alpha activation in the infected cells. AP1 transcription factors binding to the RTA promoter in electrophoretic mobility shift assays were readily detected in the infected cell nuclear extracts which were significantly reduced by ERK inhibition. Together, these results suggest that very early during de novo infection, KSHV induces the ERK1/2 to modulate the initiation of viral gene expression and host cell genes, which further supports our hypothesis that beside the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with host cell receptor(s) create an appropriate intracellular environment facilitating infection.

It is well established that Ha-ras and c-myc genes collaborate in promoting transformation, tumor progression, and metastasis. However, the precise mechanism underlying this cooperation remains unclear. In the present study, we document that astrocyte elevated gene-1 (AEG-1) is a downstream target molecule of Ha-ras and c-myc, mediating their tumor-promoting effects. AEG-1 expression is elevated in diverse neoplastic states, it cooperates with Ha-ras to promote transformation, and its overexpression augments invasion of transformed cells, demonstrating its functional involvement in Ha-ras-mediated tumorigenesis. We now document that AEG-1 expression is markedly induced by oncogenic Ha-ras, activating the phosphatidylinositol 3-kinase signaling pathway that augments binding of c-Myc to key E-box elements in the AEG-1 promoter, thereby regulating AEG-1 transcription. In addition, Ha-ras-mediated colony formation is inhibited by AEG-1 siRNA. This is a demonstration that Ha-ras activation of a tumor-promoting gene is regulated directly by c-Myc DNA binding via phosphatidylinositol 3-kinase signaling, thus revealing a previously uncharacterized mechanism of Ha-ras-mediated oncogenesis through AEG-1.

Ectopic expression of the c-Myc oncoprotein prevents cell cycle arrest in response to growth-inhibitory signals, differentiation stimuli, or mitogen withdrawal. Moreover, Myc activation in quiescent cells is sufficient to induce cell cycle entry in the absence of growth factors. Thus, Myc transduces a potent mitogenic stimulus but, concomitantly, induces apoptosis in the absence of survival factors. We review here recent progress in our understanding of the molecular mechanisms linking Myc activity to cell cycle control. Myc is a positive regulator of G1-specific cyclin-dependent kinases (CDKs) and, in particular, of cyclin E/CDK2 complexes. Cyclin D/CDK4 and CDK6 may conceivably also be activated by Myc, but the circumstances in which this occurs remain to be explored. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27Kip1 and probably also of p21Cip1 and p57Kip2, (2) induction of the CDK-activating phosphatase Cdc25A and (3) - in an ill understood and most likely indirect way - deregulation of cyclin E expression. Constitutive expression of either Myc or cyclin E can prevent growth arrest by p16INK4a (an inhibitor of cyclin D/CDK4, but not of cyclin E/CDK2). In cells, p16INK4a inhibits phosphorylation, and thus induces activation of the Retinoblastoma-family proteins (pRb, p107 and p130). Surprisingly, this effect of p16 is not altered in the presence of Myc or cyclin E. Thus, Myc and cyclin E/CDK2 activity unlink activation of p16 and pRb from growth arrest. Finally, Myc may itself be a functional target of cyclin D/CDK4 through its direct interaction with p107. We discuss how the effects of Myc on cell cycle control may relate to its oncogenic activity, and in particular to its ability to cooperate with activated Ras oncoproteins.

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using alpha-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression.

Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA, and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis, and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.

MYCN amplification is a common feature of aggressive tumour biology in neuroblastoma. The MYCN transcription factor has been demonstrated to induce or repress expression of numerous genes. MicroRNAs (miRNA) are a recently discovered class of short RNAs that repress translation and promote mRNA degradation by sequence-specific interaction with mRNA. Here, we sought to analyse the role of MYCN in regulation of miRNA expression. Using a miRNA microarray containing 384 different miRNAs and a set of 160 miRNA real-time PCR assays to validate the microarray results, 7 miRNAs were identified that are induced by MYCN in vitro and are upregulated in primary neuroblastomas with MYCN amplification. Three of the seven miRNAs belong to the miR-106a and miR-17 clusters, which have previously been shown to be regulated by c-Myc. The miR-17-92 polycistron also acts as an oncogene in haematopoietic progenitor cells. We show here that miR-221 is also induced by MYCN in neuroblastoma. Previous studies have reported miR-221 to be overexpressed in several other cancer entities, but its regulation has never before been associated with Myc. We present evidence of miRNA dysregulation in neuroblastoma. Additionally, we report miRNA induction to be a new mechanism of gene expression downregulation by MYCN.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

We revisited the cytogenetic alterations of the cervical adenocarcinoma cell line HeLa through the use of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH). SKY analysis unequivocally characterized all abnormal chromosomes. Chromosomal breakpoints were primarily assigned by simultaneous assessment of SKY painted chromosomes and inverted 4,6-diamidino2-phenylindole banding from the same cell. Twenty clonally abnormal chromosomes were found. Comparison with previously reported HeLa G-banding karyotypes revealed a remarkably stable cytogenetic constitution because 18 of 20 markers that were found were present before. The classification of 12 markers was refined in this study. Our assignment of the remaining six markers was consistent with those described in the literature. The CGH map of chromosomal copy number gains and losses strikingly matched the SKY results and was, in a few instances, decisive for assigning breakpoints. The combined use of molecular cytogenetic methods SKY, CGH, and FISH with site-specific probes, in addition to inverted 4,6-diamidino-2-phenylindole or conventional G-banding analysis, provides the means to fully assess the genomic abnormalities in cancer cells. Human papillomaviruses (HPVs) are frequently integrated into the cellular DNA in cervical cancers. We mapped by FISH five HPV18 integration sites: three on normal chromosomes 8 at 8q24 and two on derivative chromosomes, der(5)t(5;22;8)(qll;q11q13;q24) and der(22)t(8; 22)(q24;q13), which have chromosome 8q24 material. An 8q24 copy number increase was detected by CGH. Dual-color FISH with a c-MYC probe mapping to 8q24 revealed colocalization with HPV18 at all integration sites, indicating that dispersion and amplification of the c-MYC gene sequences occurred after and was most likely triggered by the viral insertion at a single integration site. Numerical and structural chromosomal aberrations identified by SKY, genomic imbalances detected by CGH, as well as FISH localization of HPV18 integration at the c-MYC locus in HeLa cells are common and representative for advanced stage cervical cell carcinomas. The HeLa genome has been remarkably stable after years of continuous cultivation; therefore, the genetic alterations detected may have been present in the primary tumor and reflect events that are relevant to the development of cervical cancer.

Mutations which disrupt the regulation or expression level of the c-myc gene are among the most common found in human and animal cancers (reviewed in ref. Cole, 1986; Henriksson and Luscher, 1996; Marcu et al., 1992). Ectopic expression studies define numerous biological activities of the c-myc gene, including transformation, immortalization, blockage of cell differentiation and induction of apoptosis (Askew et al., 1991; Cole, 1986; Evan and Littlewood, 1993; Freytag et al., 1990; Henriksson and Luscher, 1996; Marcu et al., 1992). Furthermore, c-myc is required for efficient progression through the cell cycle (Goruppi et al., 1994; Prochownik et al., 1988; Yokoyama and Imamoto, 1987), although recent studies indicate that it is not absolutely essential (Mateyak et al., 1997). This fascinating array of biological activities makes the c-myc gene one of the most intriguing oncogenes and presents the challenging question of how a single gene can manifest so many different effects. The c-Myc protein exhibits sequence-specific DNA binding when dimerized with its partner Max, and DNA binding is mediated through the basic region, which recognizes the core sequence CACGTG (Berberich et al., 1992; Blackwell et al., 1993; Blackwood and Eisenman, 1991; Prendergast and Ziff, 1991; Prendergast et al., 1991), but exhibits somewhat higher affinity for the more extended sequence ACCACGTGGT (Berberich et al., 1992; Blackwell et al., 1993; Halazonetis and Kandil, 1991). There are three closely related Myc family proteins (c-Myc, N-Myc and L-Myc), each with documented oncogenic potential (Birrer et al., 1988; Schwab et al., 1985; Yancopoulos et al., 1985) and similar DNA binding properties (Mukherjee et al., 1992). For simplicity, we will use the term Myc to refer to all three proteins, but delineate any distinct activities where they apply. The goal of this review is to discuss Myc as a transcriptional activator and critically evaluate the evidence for the transactivation of specific target genes as direct downstream effectors. Since excellent comprehensive reviews on Myc have been published recently (Facchini and Penn, 1998; Henriksson and Luscher, 1996), we will focus on the latest observations that offer mechanistic insight into transactivation and oncogenic transformation.

Infection of cattle with the protozoan parasite Theileria parva results in a fatal lymphoproliferative syndrome that is associated with the overexpression of casein kinase II. The role of this enzyme in the pathogenesis of lymphoproliferative disorders was investigated by expressing the catalytic subunit in lymphocytes of transgenic mice. Adult transgenic mice displayed a stochastic propensity to develop lymphoma; co-expression of a c-myc transgene in addition to casein kinase II resulted in neonatal leukemia. Thus, the casein kinase II gene can serve as an oncogene, and its dysregulated expression is capable of transforming lymphocytes in a two-step pathway with c-myc.

Prostate cancer is clinically heterogeneous, ranging from indolent to lethal disease. Expression profiling previously defined three subtypes of prostate cancer, one (subtype-1) linked to clinically favorable behavior, and the others (subtypes-2 and -3) linked with a more aggressive form of the disease. To explore disease heterogeneity at the genomic level, we carried out array-based comparative genomic hybridization (array CGH) on 64 prostate tumor specimens, including 55 primary tumors and 9 pelvic lymph node metastases. Unsupervised cluster analysis of DNA copy number alterations (CNA) identified recurrent aberrations, including a 6q15-deletion group associated with subtype-1 gene expression patterns and decreased tumor recurrence. Supervised analysis further disclosed distinct patterns of CNA among gene-expression subtypes, where subtype-1 tumors exhibited characteristic deletions at 5q21 and 6q15, and subtype-2 cases harbored deletions at 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion). Lymph node metastases, predominantly subtype-3, displayed overall higher frequencies of CNA, and in particular gains at 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23. Our findings reveal that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating prostate cancer biology and explain in part the clinical heterogeneity of the disease.

TIP60 was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat. As a consequence, the role of TIP60 in transcriptional regulation has been investigated intensively. Recent data suggest that TIP60 has more divergent functions than originally thought and roles for TIP60 in many processes, such as cellular signalling, DNA damage repair, cell cycle and checkpoint control and apoptosis are emerging. TIP60 is a tightly regulated transcriptional coregulator, acting in a large multiprotein complex for a range of transcription factors including androgen receptor, Myc, STAT3, NF-kappaB, E2F1 and p53. This usually involves recruitment of TIP60 acetyltransferase activities to chromatin. Additionally, in response to DNA double strand breaks, TIP60 is recruited to DNA lesions where it participates both in the initial as well as the final stages of repair. Here, we describe how TIP60 is a multifunctional enzyme involved in multiple nuclear transactions.

Phorbol ester tumor promoters, such as phorbol 12-myristate 13-acetate (PMA), are potent activators of extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase (SAPK), and p38 mitogen-activated protein kinase (MAPK) in U937 human leukemic cells. These kinases are regulated by the reversible dual phosphorylation of conserved threonine and tyrosine residues. The dual specificity protein phosphatase MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate and inactivate ERK2, SAPK, and p38 MAPK in transient transfection studies. Here we demonstrate that PMA treatment induces MKP-1 protein expression in U937 cells, which is detectable within 30 min with maximal levels attained after 4 h. This time course coincides with the rapid inactivation of PMA-induced SAPK activity, but not ERK2 phosphorylation, which remains elevated for up to 6 h. To examine directly the role of MKP-1 in the regulation of these protein kinases in vivo, we established a U937 cell line that conditionally expresses MKP-1 from the human metallothionein IIa promoter. Conditional expression of MKP-1 inhibited PMA-induced ERK2, SAPK, and p38 MAPK activity. By titrating the levels of MKP-1 expression from the human metallothionein IIa promoter, however, it was found that p38 MAPK and SAPK were much more sensitive to inhibition by MKP-1 than ERK2. This differential substrate specificity of MKP-1 can be functionally extended to nuclear transcriptional events in that PMA-induced c-Jun transcriptional activity was more sensitive to inhibition by MKP-1 than either Elk-1 or c-Myc. Conditional expression of MKP-1 also abolished the induction of endogenous MKP-1 protein expression in response to PMA treatment. This negative feedback regulatory mechanism is likely due to MKP-1-mediated inhibition of ERK2, as studies utilizing the MEK1/2 inhibitor PD98059 suggest that ERK2 activation is required for PMA-induced MKP-1 expression. These findings suggest that ERK2-mediated induction of MKP-1 may play an important role in preferentially attenuating signaling through the p38 MAPK and SAPK signal transduction pathways.

To elucidate the contribution of the N-Myc protein to neuroblastomas we have used a synthetic inducible expression system on the basis of the tetracycline repressor of E coli to reversibly express N-myc in a human neuroblastoma cell line in which expression of endogenous N-myc is barely detectable. Like the c-Myc protein, N-Myc up-regulates the expression of both alpha-prothymosin and ornithine decarboxylase. Induction of N-myc increases both the rate of DNA-synthesis and the proliferation rate, and shortens the G1 phase of the cell cycle. A comparison of cell populations in which the presence of N-Myc protein was restricted to different parts of G(zero)/G1 revealed that N-Myc is rate-limiting for cell cycle progression during the first 5 h after serum stimulation of quiescent cells providing direct evidence that Myc-proteins act early after mitogenic stimulation of quiescent cells.

The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a>> 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

Beta-catenin-dependent or canonical Wnt signals are fundamental in animal development and tumor progression. Using Xenopus laevis, we report that the BTB/POZ zinc finger family member Kaiso directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1, and c-Myc) in conjunction with TCF/LEF (TCF). Analogous to beta-catenin relief of TCF repressive activity, we show that p120-catenin relieves Kaiso-mediated repression of Siamois. Furthermore, Kaiso and TCF coassociate, and combined Kaiso and TCF derepression results in pronounced Siamois expression and increased beta-catenin coprecipitation with the Siamois promoter. The functional interdependency is underlined by Kaiso suppression of beta-catenin-induced axis duplication and by TCF-3 rescue of Kaiso depletion phenotypes. These studies point to convergence of parallel p120-catenin/Kaiso and beta-catenin/TCF signaling pathways to regulate gene expression in vertebrate development and possibly carcinogenesis.

Astrocyte elevated gene-1 (AEG-1) displays oncogenic properties. Its expression is elevated in diverse neoplastic states and it cooperates with Ha-ras to promote cellular transformation. Overexpression of AEG-1 augments invasion and anchorage-independent growth of transformed cells, while AEG-1 siRNA inhibits Ha-ras-mediated colony formation, supporting a potential functional role in tumorigenesis. Additionally, oncogenic Ha-ras induces AEG-1 expression through the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In the present study, we investigated whether AEG-1 could induce serum-independent cell growth, another property of oncogenes. Overexpression of AEG-1 inhibited serum starvation-induced apoptosis through activation of PI3K-Akt signaling, one of the effector pathways induced by activated Ras. AEG-1 also affected the phosphorylation state of Akt substrates that are implicated in apoptosis suppression, including glycogen synthase kinase 3beta, c-Myc, murine double minute 2, p53, p21/mda-6 and Bad. Additionally, AEG-1 blocked the activity of serum starvation-induced caspases. Taken together, these observations provide evidence that AEG-1 is an oncogene cooperating with Ha-ras as well as functioning as a downstream target gene of Ha-ras and may perform a central role in Ha-ras-mediated carcinogenesis. Activation of survival pathways may be one mechanism by which AEG-1 exerts its oncogenic properties.

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. In this study, we report the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic "oncomiR" and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)-dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. Our findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer.

We describe a signaling pathway that mediates salt stress responses in Arabidopsis. The response is mechanistically related to endoplasmic reticulum (ER) stress responses described in mammalian systems. Such responses involve processing and relocation to the nucleus of ER membrane-associated transcription factors to activate stress response genes. The salt stress response in Arabidopsis requires a subtilisin-like serine protease (AtS1P), related to mammalian S1P and a membrane-localized b-ZIP transcription factor, AtbZIP17, a predicted type-II membrane protein with a canonical S1P cleavage site on its lumen-facing side and a b-ZIP domain on its cytoplasmic side. In response to salt stress, it was found that myc-tagged AtbZIP17 was cleaved in an AtS1P-dependent process. To show that AtS1P directly targets AtbZIP17, cleavage was also demonstrated in an in vitro pull-down assay with agarose bead-immobilized AtS1P. Under salt stress conditions, the N-terminal fragment of AtbZIP17 tagged with GFP was translocated to the nucleus. The N-terminal fragment bearing the bZIP DNA binding domain was also found to possess transcriptional activity that functions in yeast. In Arabidopsis, AtbZIP17 activation directly or indirectly upregulated the expression of several salt stress response genes, including the homeodomain transcription factor ATHB-7. Upregulation of these genes by salt stress was blocked by T-DNA insertion mutations in AtS1P and AtbZIP17. Thus, salt stress induces a signaling cascade involving the processing of AtbZIP17, its translocation to the nucleus and the upregulation of salt stress genes.

The etiology of small cell lung cancer (SCLC) is strongly tied to cigarette smoking, and now there is considerable information concerning molecular abnormalities involved in the pathogenesis of SCLC. Autocrine growth factors such as neuroendocrine regulatory peptides (eg, bombesin/gastrin-releasing peptide) are prominent in SCLC. Dominant oncogenes of the Myc family are frequently overexpressed in both SCLC and non-small cell lung cancer (NSCLC), while the K-RAS oncogene is never mutated in SCLC but it is in 30% of NSCLCs. The most frequent genetic abnormalities involve tumor suppressor genes (TSGs). The TSG p53 is mutated in more than 90% of SCLCs and more than 50% of NSCLCs; the retinoblastoma TSG is inactivated in over 90% of SCLC but only 15% of NSCLCs, and p16, the other component of the retinoblastoma/p16 pathway, is almost never abnormal in SCLC but is inactivated in more than 50% of NSCLCs. The FHIT TSG is inactivated in 50% to 70% of all lung cancers. Recently, we completed a genome-wide allelotyping study using approximately 400 polymorphic markers distributed at around 10 cM resolution across the human genome comparing SCLCs and NSCLCs, looking for all possible TSG sites by loss of heterozygosity. We found that, on average, 17 loci showed loss of heterozygosity in individual SCLCs and 22 for NSCLC, with an average size of loss of 50 to 60 cM, and an average frequency of microsatellite abnormalities of five per tumor. There were 22 different "hot spots" for loss of heterozygosity, 13 with a preference for SCLC, seven for NSCLC, and two affecting both. This provides clear evidence on a genome-wide scale that SCLC and NSCLC differ significantly in the TSGs that are inactivated during their pathogenesis. Acquired hypermethylation of the promoter region of key genes has become one of the most common mechanisms that tumors use to inactivate the function of tumor suppressor and other genes. We recently completed a study of tumor-acquired promoter hypermethylation for nine genes (p16, DAPK, MGMT, GSTP1, RAR beta, FHIT, ECAD, p14ARF, and TIMP1). We found differences in the frequency of RAR beta methylation (70% for SCLC and 40% for NSCLCs). Finally, we looked at the bronchial epithelium accompanying SCLC and NSCLC for the occurrence of clonal alterations using precise laser capture microdissection with subsequent allelotyping for polymorphic markers. In NSCLC, we frequently find clones of cells with molecular abnormalities in histologically affected epithelium (eg, carcinoma in situ, dysplasia, hyperplasia) and occasionally in normal-appearing epithelium in the cases of current or former smokers. In SCLC these histologic preneoplastic changes were minimal. However, in studies of histologically normal respiratory epithelium, we found a several-fold increased rate of allele loss in SCLC compared with NSCLC patients. Thus, the smoking-damaged histologically normal epithelium associated with SCLC appeared genetically scrambled and has incurred significantly more damage than the epithelium accompanying NSCLCs. We conclude that SCLC and NSCLCs do not differ significantly in the number of genetic alterations that occur. However, SCLCs do differ significantly from NSCLCs in the specific genetic alterations that occur. In addition, smoking-damaged bronchial epithelium accompanying SCLCs appears to have undergone significantly more acquired genetic damage than that accompanying NSCLCs. Future studies need to identify the specific genes involved at these multiple sites and determine if these provide new tools for early molecular detection and monitoring of chemoprevention efforts, and serve as specific targets for developing new therapies. Semin Oncol 28 (suppl 4):3-13.

OBJECTIVE

Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors.

METHODS

BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2D(b) to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples.

RESULTS

We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model.

CONCLUSIONS

These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell-based immunotherapy for the treatment of patients with melanoma.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.